JP2004534508A - Mice deficient in corticotropin releasing factor receptor 2 and their use - Google Patents

Abstract

The present invention provides a transgenic mouse lacking corticotropin releasing factor receptor 2 (CRFR2). Mice lacking CRFR1 show reduced anxiety behavior and reduced stress response. In contrast, CRFR2 deficient mutant mice show hyperresponse to stress and increased anxiety behavior. They are useful for studying the physiology of anxiety, depression, and the HPA axis. CRFR2 deletion mutant mice showed increased angiogenesis in all tissues examined. Thus, CRFR2 antagonists can be used to promote angiogenesis for the treatment of various conditions. In contrast, CRFR2 agonists can be used to inhibit angiogenesis. It was observed that the combination of urocortin and bFGF induced rapid hair growth.

Description

[0001]
(Federal funding)
This invention was made in part with federal government funding NIH DK-26741 and NRSA Fellowships DK09841 and DK09551. Accordingly, the federal government has certain rights in the invention.
[0002]
(Field of the Invention)
The present invention relates generally to the fields of neurobiology, endocrinology, and psychiatry. More specifically, the present invention relates to studies of anxiety and to mice lacking corticotropin releasing factor receptor 2.
[0003]
(Explanation of related technology)
Corticotropin releasing factor (CRF) is a key regulator of the hypothalamus-pituitary-adrenal (HPA) axis. In response to stress, corticotropin-releasing factor released from the hypothalamus paraventricular nucleus (PVN) activates the corticotropin-releasing factor receptor on the anterior pituitary adrenocortical stimulator, resulting in corticotropin stimulating hormone in the bloodstream. (ACTH) is released. ACTH, on the other hand, activates the ACTH receptor in the adrenal cortex, increasing the synthesis and release of glucocorticoid (1).
[0004]
The receptors for CRF, CRFR1 and CRFR2, are located throughout the CNS and periphery. CRF has a higher affinity for CRFR1 than for CRFR2, and the CRF-related peptide urocortin (UCN) binds with nearly 40-fold higher affinity than CRF and is therefore considered to be an endogenous ligand for CRFR2 ( 2). The amino acid similarity between CRFR1 and CRFR2 is about 71%, and their localization in brain and surrounding tissues is different (3-6). CRFR1 is mainly expressed in the pituitary, cerebral cortex, cerebellum, hindbrain, and olfactory bulb, whereas CRFR2 is expressed in the lateral septum, the ventromedial hypothalamus (VMH), the choroid plexus, and many peripheral sites. Seen (3,7).
[0005]
Mice lacking CRFR1 have reduced levels of hormones on the HPA axis, impaired stress response function, and reduced anxiety-like behavior (8, 9). These results are consistent with those obtained using CRFR1 specific antagonists in vitro (10-12). On the other hand, CRFR2-specific antagonists are not currently available and little has been elucidated about the physiological function of CRFR2 since it was cloned in 1995. UCN is a potential endogenous ligand for CRFR2 and has been shown to regulate feeding behavior when administered centrally (13). Because CRFR2 is localized in the ventromedial hypothalamus, a central site for regulation of eating behavior and satiety, the action of urocortin at these receptors may influence eating behavior. Furthermore, peripheral administration of urocortin is thought to be the result of action at CRFR2 in vascular endothelial cells (3,7) leading to hypertension (2,14). Therefore, CRFR2 non-expressing mutant mice were generated and analyzed to identify the developmental and physiological roles of CRFR2.
[0006]
The prior art is deficient in lacking mice lacking corticotropin releasing factor receptor 2. The present invention fulfills a long-felt need and desire in the art.
[0007]
(Summary of the Invention)
CRFR2 deficient mice have increased anxiety-like behavior and show HPA axis hypersensitivity in the stress response. Mutant mice that do not express CRFR1 and CRFR2 provide a valuable model of anxiety and depression and can further facilitate the elucidation of the molecular mechanisms underlying these diseases. Studies on the signaling pathway of corticotropin-releasing factor and its role in the treatment of anxiety and depression will provide the necessary clues required for effective treatment of these diseases.
[0008]
Accordingly, the present invention relates to a non-native transgenic mouse in which at least one allele of corticotropin releasing factor receptor 2 (CRFR2) has been disrupted, said mouse comprising a corticotropin releasing factor receptor 2 (CRFR2) from said allele. Not expressed. Preferably, the DNA sequences of exons 10, 11, and 12 of the corticotropin releasing factor receptor 2 allele have been removed. The transgenic mouse may have these DNA sequences replaced with the neomycin resistance gene cassette. The transgenic mouse may be either heterozygous or homozygous for this substitution. One embodiment of the present invention also includes the results of crossing the mouse according to the present invention with a mouse of another strain.
[0009]
Another embodiment of the present invention is the use of CRFR2-deficient mice to study anxiety or depression and to test the effects of various compounds on anxiety or depression. For example, one method of screening for a compound having anxiolytic activity is provided, comprising: a) administering the compound to a transgenic mouse according to the invention; b) treating the mouse for behaviors associated with anxiety. Testing; and c) comparing the anxiety-like behavior of said mouse to the anxiety-like behavior of a second transgenic mouse according to the present invention to which said compound is not administered. Further provided is a method of screening for compounds having depressant-relieving activity, comprising: a) administering the compound to a transgenic mouse according to the invention; b) testing said mouse for behaviors associated with depression. And c) comparing the depression-like behavior of the mouse to the depression-like behavior of the second transgenic mouse according to the present invention to which the compound is not administered.
[0010]
Yet another embodiment of the invention involves the use of CRFR2-deficient mice in a similar procedure to screen for compounds that affect blood pressure or angiogenesis.
[0011]
Yet another embodiment of the present invention provides a method for screening CRPA2-deficient mice for studying HPA axis physiology, such as one method of screening for compounds that have an effect on the response of the hypothalamus-pituitary-adrenal axis to stress. The method of use is: a) administering the compound to a transgenic mouse according to the invention; b) placing the mouse in a stress-induced state; c) stimulating corticosterone and adrenocortical of the mouse. Monitoring plasma levels of the hormone; and d) comparing said levels to the levels of transgenic mice according to the present invention that are not subjected to stress-induced conditions.
[0012]
In yet another embodiment of the invention, the mouse can be used to monitor the effects of compounds on the response of the HPA axis to stress by monitoring plasma levels of corticosterone and ACTH.
[0013]
Yet another embodiment of the present invention relates to the use of said mice in studying the effect of corticotropin releasing factor receptor 2 on other proteins such as corticotropin releasing factor and urocortin.
[0014]
Yet another embodiment of the present invention is the use of CRFR2-deficient mice to examine CRFR1 response without being hindered by the presence of CRFR2.
[0015]
Studies of CRFR2 null mutant mice revealed that deletion of the CRFR2 gene resulted in increased angiogenesis in all tissues tested. Thus, another embodiment according to the present invention is the use of a CRFR2 null mutant mouse for studying the molecular mechanisms of angiogenesis regulation.
[0016]
In yet another embodiment of the invention, angiogenesis is promoted in the target tissue by administering a CRFR2 antagonist to the tissue. One example of such an antagonist is an antisense nucleotide directed against the CRFR2 gene. The heart, brain, pituitary, gonads, kidney, fat, and gastrointestinal tract are among the tissues where such a response is achieved. This promotion of angiogenesis has been shown to be beneficial in treating infarcts, strokes and injuries.
[0017]
In yet another embodiment of the present invention, angiogenesis is inhibited in the target tissue by administration of a CRFR2 agonist such as urocortin or CRF. CRFR2 action-induced inhibition of angiogenesis can be used to treat cancer and diabetic retinopathy.
[0018]
Another embodiment according to the invention is a method of stimulating hair growth by implanting urocortin and bFGF below the area of skin where hair growth is desired, or contacting urocortin in a topical composition with the skin. It is about the method.
[0019]
(Detailed description of the invention)
In accordance with the present invention, conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art can be used. Such techniques are explained fully in the literature. See, for example, Maniatis, Fritsch & Sambrook, "Molecular Cloning: A Laboratory Manual (1982);" J. Gait ed. 1984); "Nucleic Acid Hybridization" [BD Hames & S. J. Higgins eds. (1985)]; "Transscription and Translation and [J.D. (1984)]; "Animal Cel. Culture "[R. I. Freshney, ed (1986).];" Immobilized Cells And Enzymes "[IRL Press, (1986)]; B Perbal," A Practical Guide To Molecular Cloning "see (1984).
[0020]
Thus, as used herein, the following terms shall have the definitions set out below.
[0021]
As used herein, "cDNA" refers to a copy of the DNA of a gene's mRNA transcript.
[0022]
As used herein, the term "screening a library" is used to determine, under appropriate conditions, whether a sequence complementary to a probe present in a particular DNA library is present. Refers to the process using labeled probes. Further, "screening the library" can be performed by PCR.
[0023]
As used herein, the term "PCR" refers to the polymerase chain reaction that is the subject of Mullis U.S. Patent Nos. 4,683,195 and 4,683,202 and other improvements well known in the art. I have.
[0024]
The amino acids mentioned herein are preferably the "L" isomer. However, the residues of the "D" isomer can be replaced with any L amino acid residue, as long as the desired functional properties of immunoglobulin binding are retained by the polypeptide. NH₂Refers to the free amino group present at the amino terminus of the polypeptide. COOH is a free carboxy group present at the carboxy terminus of a polypeptide. Glossary of standard polypeptide terminology, Biol. Chem. , 243: 3552-59 (1969), and abbreviations for amino acid residues are well known in the art.
[0025]
In the present specification, all amino acid residue sequences are represented in a conventional manner in which the direction from left to right is from the amino terminal to the carboxy terminal. Further, a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond of one or more amino acid residues to another sequence.
[0026]
A "replicon" is any genetic element (eg, a plasmid, chromosome, virus) that functions as a self-sustaining unit of DNA replication in vivo, ie, capable of replication under its own control.
[0027]
A "vector" is a replicon, such as a plasmid, phage, or cosmid, to which another DNA segment has been attached and causes the attached segment to replicate.
[0028]
The term “DNA molecule” refers to a polymer in the form of a single-stranded or double-stranded helical deoxyribonucleotide (adenine, guanine, thymine, or cytosine) polymer. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, the term includes double-stranded DNA found in, among others, linear DNA molecules (eg, restriction fragments), viruses, plasmids, and chromosomes. The discussion of the structure herein follows standard practice of providing only sequences in the 5 'to 3' direction along the non-transcribed strand of DNA (e.g., a strand having a sequence homologous to mRNA).
[0029]
“Replication origin” refers to a DNA sequence involved in DNA synthesis.
[0030]
A "coding sequence" of DNA is a double-stranded DNA sequence that is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5 '(amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. Coding sequences include, but are not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (eg, mammalian) DNA, and even synthetic DNA sequences. The polyadenylation signal and transcription termination sequence are usually located 3 'to the coding sequence.
[0031]
Transcription and translation control sequences are DNA control sequences that provide expression of the coding sequence in the host cell, such as promoters, enhancers, polyadenylation signals, terminators, and the like.
[0032]
A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3 'direction) coding sequence. For the purposes of clarifying the present invention, the promoter sequence is bound at its 3 'end by a transcription initiation site and has a minimal number of bases or components necessary to initiate transcription at a detectable level above background. It is extended upstream (5 'direction) to include the number. Within the promoter sequence will also be found a transcription initiation site, and a protein binding region (consensus sequence) responsible for RNA polymerase binding. Eukaryotic promoters often, but not always, contain "TATA" boxes and "CAT" boxes. Prokaryotic promoters contain SD sequences in addition to the -10 and -35 consensus sequences.
[0033]
An "expression control sequence" is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence. When RNA polymerase transcribes a coding sequence into mRNA, the coding sequence is "under control" of transcriptional and translational regulatory sequences in a cell, and is subsequently translated into a protein encoded by the coding sequence.
[0034]
The “signal sequence” is near the coding sequence. This sequence encodes a signal peptide at the N-terminus of the polypeptide, which directs the polypeptide to the cell surface or directs the polypeptide to the host cell for secretion into the medium, wherein the signal peptide is a protein Are dissociated by host cells before detaching the cells. Signal sequences are found in connection with various proteins that are native to prokaryotes and eukaryotes.
[0035]
"Oligonucleotide" is defined as a molecule consisting of two or more, more preferably three or more ribonucleotides, as used herein for a probe according to the present invention. The exact size will depend on many factors that depend on the ultimate function and utilization of the oligonucleotide.
[0036]
"Primer", as used herein, refers to a primer extension complementary to a nucleic acid strand, whether naturally occurring, such as a purified restriction digest, or artificially generated. An oligonucleotide that can function as a starting point for synthesis at the appropriate temperature and pH under the conditions that induce the synthesis of the product, ie, in the presence of nucleotides and derivatives such as DNA polymerase. Primers can be single-stranded or double-stranded, but must be long enough to prime the synthesis of preferred extension products in the presence of the derivative. The exact length of the primer will depend on many factors, including temperature, source of primer and use. For example, when used in analysis, depending on the complexity of the target sequence, oligonucleotide primers typically contain 15 to 25 or more nucleotides, and sometimes contain fewer nucleotides.
Primers herein are directed to different strands of a particular target DNA sequence.
[0037]
It is selected to be "substantially" complementary. This means that the primer must be sufficiently complementary to hybridize with its respective strand. Thus, the primer sequence need not accurately reflect the template sequence. For example, a non-complementary nucleotide fragment may be attached to the 5 'end of a primer, and the remainder of the primer sequence may be complementary to that strand. Also, if the primer sequence is sufficiently complementary to, or hybridizes to, the sequence, thereby forming a template for synthesis of the extension product, non-complementary bases or longer sequences may be used. Scattered in primers.
[0038]
As used herein, "restriction endonucleases" and "restriction enzymes" refer to all enzymes that cut double-stranded DNA at or near a specific nucleotide sequence.
[0039]
A cell is "transformed" by exogenous or heterologous DNA when such DNA has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. For example, in prokaryotes, yeast, and mammalian cells, the transforming DNA may be maintained on an episomal factor such as a plasmid. With respect to eukaryotic cells, stably transformed cells have the transforming DNA integrated into the chromosome such that they are inherited by daughter cells via chromosome replication. This stability is demonstrated by the ability of eukaryotic cells to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A "clone" is a group of cells derived from a single cell or a mitotic progenitor. A “cell line” is a clone of a primary cell that can grow stably for many generations in vitro.
[0040]
Generally, an expression vector containing a promoter sequence that promotes efficient transcription of the inserted DNA fragment is used in connection with the host. The expression vector usually also contains an origin of replication, a promoter, a terminator, as well as specific genes capable of providing phenotypic selection in transformed cells. The transformed host can be fermented and cultured according to methods well known in the art to achieve optimal growth of the cells.
[0041]
Methods well known to those skilled in the art can be used to create expression vectors containing the appropriate transcription and translation control signals. For example, see Sambrook et al. , "Molecular Cloning: A Laboratory Manual (2nd Ed.), Cold Spring Harbor Press, NY. One gene and its transcription control sequence, if its transcription control sequence is the gene's An effective control of transcription is defined as “operably linked.” Vectors according to the present invention include plasmid vectors and viral vectors.
[0042]
The present invention relates to CRFR2 deficient mice, which were created to identify the developmental and physiological role of CRFR2 in anxiety and HPA axis circuitry. This was done by deleting exons 10, 11, and 12 of the corticotropin releasing factor receptor 2 allele. In the present invention, these nucleotide sequences were replaced with a neomycin resistance gene cassette. The mouse may be either heterozygous or homozygous for its CRFR2 deletion and may be bred to another strain of mice.
[0043]
The present invention also relates to the use of CRFR2-deficient mice in studies of anxiety or depression, such as methods for testing compounds that act to alleviate anxiety or depression. Compounds that affect blood pressure or angiogenesis can also be screened using CRFR2-deficient mice.
[0044]
The invention also relates to the use of CRFR2-deficient mice in studies of the molecular physiology of the hypothalamus-pituitary-adrenal (HPA) axis. The mice can also be used to test the effect of the compound on the response of the HPA axis to stress.
[0045]
The invention also relates to the use of said transgenic mice to study the molecular function of corticotropin releasing factor receptor 2 on corticotropin releasing factor, corticotropin releasing factor receptor 1, urocortin and other CRF and urocortin receptors.
[0046]
In addition, the present invention can be used to study the response and action of CRFR1 in the negative environment of CRFR2 (CRFR2 negative environment). In this sense, the CRFR1 response can be studied unhindered by the modulation of CRFR2.
[0047]
The present invention also relates to the use of CRFR2 non-expressing mutant mice for molecular regulation of angiogenesis.
[0048]
The invention also relates to a method of promoting angiogenesis by administering a CRFR2 antagonist to a target tissue. One way in which this is achieved is through the use of antisense nucleotides directed against the CRFR2 gene. Tissues where such a response is achieved include the heart, brain, pituitary, gonads, kidney, fat, and gastrointestinal tract. The present invention has been shown to be beneficial in promoting angiogenesis after infarction, stroke, and injury.
[0049]
The present invention also relates to a method of inhibiting angiogenesis by administering a CRFR2 agonist to a target tissue, such as the heart, brain, pituitary, gonad, kidney, fat, and gastrointestinal tract. CRFR2 agonists include urocortin or CRF. Cancer and diabetic retinopathy are examples of diseases responsive to CRFR2 agonist-induced inhibition of angiogenesis.
[0050]
The present invention relates to a method of promoting hair growth comprising the step of contacting urocortin with the skin on the skin area where hair growth is desired. In one embodiment, the urocortin is implanted under the skin. While urocortin alone may be useful, bFGF may be administered before, after, or simultaneously with urocortin administration. Urocortin may be included in the composition along with bFGF.
[0051]
【Example】
The following examples are given for the purpose of illustrating various embodiments according to the present invention and are not meant to limit the invention in any way.
[0052]
Example 1
Production of CRFR2-deficient mice
One genomic clone DNA containing the CRFR2 locus was isolated from a mouse strain 129 genomic DNA library to generate CRFR2 non-expressing mutant mice. From this clone, a target vector was prepared in which exons 10, 11, and 12 of the CRFR2 gene encoding from the beginning of transmembrane region 5 to the end of transmembrane region 7 were replaced with a neomycin resistance gene cassette (FIG. 1A). The resulting plasmid DNA was linearized with Not I and electroporated into J1 embryonic stem (ES) cells as described previously (8). After selection in 0.2 mg / ml G418 (active) for 7 to 9 days, neomycin resistant clones were individually selected and screened for the presence of the disrupted CRFR2 allele by Southern blot analysis.
[0053]
Positive ES clones were injected into C57 BL / 6 blastocysts to generate chimeric mice. Chimeric mouse males were bred to C57 BL / 6 females and germline transmission of the disrupted allele was determined by Southern blot analysis of tail DNA from F1 offspring with Agouti coat color (Fig. 1B).
[0054]
Example 2
Analysis of CRFR1 and CRFR2 expression in CRFR2-deficient mice
To determine whether the targeted deletion resulted in a non-expressing mutant of the CRFR2 gene, receptor autoradiography was performed on brain sections from wild-type controls and mutant mice.
[0055]
The slide on which the 20 μm sectioned brain tissue was set was thawed at room temperature, and washed twice with 50 mM Tris buffer (pH 7.4) at room temperature for 10 minutes. Thereafter, the section was subjected to 50 mM Tris (pH 7.4),¹²⁵I-sorbazine, 10 mM MgCl₂, 0.1% BSA, and 0.05% bacitracin for 60 minutes at room temperature.¹²⁵Non-specific binding was demonstrated on adjacent sections exposed to both I-sorbazine and 1 μm cold sorbazine. After the culture period, the slides were washed twice with 50 mM Tris buffer plus 0.01% Triton X-100 at 4C for 5 minutes. The slides were immediately immersed in pure water, dried and juxtaposed for 3 days to form a thin film.
[0056]
In mutant mice, no binding in the brain region specific for CRFR2 (outer septum) was detected, but binding to CRFR1 was maintained in the cortex (FIG. 1C). These results indicate that CRFR2 gene disruption led to non-expressed mutations in these mice. Mutant mice were fertile and transmitted mutant alleles according to Mendel's law.
[0057]
Example 3
Histological analysis of CRFR2-deficient mice
To determine if HPA axis development was impaired in CRFR2 non-mutant mutant mice, the pituitary and adrenal glands of 10-12 week old male mice were sectioned and stained with hematoxylin and eosin (H & E). That is, mice were perfused with 4% paraformaldehyde (PFA). Tissues were removed, post-fixed overnight at 4C, and cryoprotected with 30% sucrose in PBS. Tissues were sectioned to a thickness of 12 μm and stained with hematoxylin and eosin. The results showed no clear differences in structure or cell type (FIGS. 1D, 1E).
[0058]
In addition, sections of the pituitary were stained with an anti-ACTH antibody. Pituitaries were sectioned, post-fixed with 4% PFA for 5 minutes, rinsed with PBS, and stained with ACTH antibody as described previously (6). No qualitative difference was observed between wild-type and mutant adrenal cortex stimulating factors (FIG. 1E).
[0059]
Example 4
Determination of corticosterone and ACTH levels in CRFR2-deficient mice
Plasma was obtained from individually bred 10-12 week old male mice for corticosterone and ACTH analysis. Samples were collected from retroorbital blood from unanesthetized mice with cage disruption within 30 seconds. A basal AM sample was collected at 7:00 AM. A basal PM sample was collected at 5:00 PM. Radioimmunoassay kits were weighed identically and used 5 μl of plasma in a corticosterone assay (ICN Biomedicals, Dosta Mesa, Calif.) And an ACTH assay (Nichols Institute Diagnostics, San Juan Capistrano, San J. CA.). 50 μl of plasma was used. Standard basal levels of ACTH and corticosterone were found in mutant and control mice (FIGS. 2A, 2B), consistent with the finding that ACTH levels were not affected in the brain.
[0060]
Example 5
Effect of stress on HPA axis response in CRFR2-deficient mice
To examine the response of the HPA axis to stress, mice were placed under physically restrained stress for progressively longer periods of time. Blood samples were taken immediately after any 2, 5, or 10 minute restraint stress in a 50 ml conical tube (plastic conical tube with the bottom removed). Plasma samples were immediately centrifuged and stored at −20 ° C. until evaluation.
[0061]
ACTH levels in the mutant mice increased significantly and peaked only 2 minutes after restraint stress (FIG. 2C). On the other hand, the ACTH level in the control mice reached a maximum 10 minutes after restraint. Similarly, the levels of corticosterone in mutant mice increased significantly 2 minutes after restraint, while levels in control mice increased 5 minutes after stress (FIG. 2D). These results revealed a hypersensitive response of the HPA axis to stress in mutant mice.
[0062]
Example 6
CRFR2-deficient mice are hypersensitive to food deprivation
Since CRFR2 is abundant in VMH and previous studies have shown an anorectic effect of icv urocortin (13), basal feeding and weight gain were measured in mutant and wild-type littermates.
[0063]
Basal feeding was measured in individually bred male littermates from 12 to 16 weeks of age. The mice and their dietary pellets were weighed daily at 9:00. For a food deprivation experiment, a control group and mutant litters were individually bred, and their basal food intake and body weight were determined. Mice were deprived of food for 24 hours starting at 12:00, but had free access to water. After the food deprivation period, mice were weighed and given pre-weighed food pellets. Thereafter, the weight of the diet pellet was measured every two hours until the light was turned off (18:00). The diet pellets and the mice were weighed again the next morning. Weight loss during food deprivation and increase in total food consumption and body weight 24 hours after food deprivation were recorded.
[0064]
Male basal feeding and weight gain of CRFR2 non-expressing mutant (mut) mice were similar to wild-type (wt) fetuses (24-hour dietary consumption wt = 4.3 ± 0.24 g, mut = 4. 6 ± 0.23 g; body weight wt = 21.7 ± 0.66 g, mut = 21.2 ± 0.50 g; n = 10, mean ± sem).
To determine whether the stressful stimulus alters the dietary intake of mutant mice, the control group and the mutant mice were deprived of food for 24 hours and then re-fed, and the change in food intake and body weight of the mice was measured. did. The results of food deprivation showed a significant decrease in food intake of mutant mice after 24 hours of food deprivation (FIG. 3A). Mutant mice consumed 75% of the diet level of the wild-type during the 24-hour period after food deprivation. However, the body weight of the mutant and wild species did not differ significantly after food deprivation or refeeding (3B).
[0065]
Example 7
Evaluation of anxiety-like behavior in CRFR2-deficient mice in the elevated plus maze
Since CRFR1 mutant mice displayed behavior similar to anxiety (8), CRFR2 non-expressing mutant mice were analyzed in a similar experiment. Control groups and mutant mice were evaluated using the elevated plus maze (EPM). Male mice between 22 and 24 weeks of age were used in this experiment. Littermate wild-type mice were used as a control group. The mice were housed in groups and maintained in regular light / dark conditions (lights on at 6:00 AM, lights off at 6:00 PM) and were handled every other day one week prior to the experiment.
[0066]
The cross maze device was made of black plexiglas, had two open arms (30 x 5 cm) and a closed arm of the same size, and had a wall 30 cm high. The device was elevated 30 cm above the floor. The arms were connected to a central square (5 × 5 cm), thereby forming a maze device in a cross shape. A 25 watt light was placed above the device and the open arm light intensity was 6 lux. All experiments were performed during the light phase of the light-dark cycle. Mice were allowed to acclimate to laboratory conditions for one hour before behavioral experiments, and test mice were individually subjected to a five-minute experiment once.
[0067]
The experiment was started with each mouse placed on a center platform facing the open arm. Behavior recorded was the number of open and closed arm entries and the time spent in various parts of the maze. Entry into the arm was defined as all four legs entering the arm. Entry into the closed arm was considered an indicator of locomotor activity in a cross-shaped maze. The camera installed above the device allowed the behavior of the mouse to be observed on a video monitor in the adjacent room. At the end of the experiment, the number of entries into the open arm and the duration were expressed as percentages of the number of entries into all arms and the duration of the experiment, respectively. Results are expressed as mean ± standard error of the mean. Behavioral parameters obtained from the EPA test were analyzed using the Student's t-test.
[0068]
As a result, it was shown that the CRFR2 non-expressing mutant mouse had a shorter staying time in the open arm of the cruciform maze device and a smaller number of entries than the wild-type control group mouse. Both the entry rate into the open arm [t (12) = 2.684; p <0.02] and the staying time rate in the open arm [t (12) = 3.524; p <0.005] (FIG. 4A). An increase in anxiety-like behavior is due to the overall behavior of the closed arm [t (12) = 0.469; p = 0.64] and entry to all arms [t (12) = 0.904; p = 0.38] was not due to a change in locomotor activity, as there was no difference between the two groups (FIG. 4B). These results indicate that CRFR2 non-expressing mutant mice significantly increased anxiety-like behavior.
[0069]
Example 8
Evaluation of anxiety-like behavior in CRFR2-deficient mice in a light-dark box
The behavior of CRFR2 non-expressing mutant mice and control mice was also analyzed for behavior similar to anxiety in a light-dark box. The rectangular plexiglass box was divided into two compartments, one painted white (28.5 cm x 27.0 cm) and the other painted black (14.5 cm x 27.0 cm). The luminosity of the black compartment covered by the red Plexiglas lid was 8 lux and the luminosity of the white compartment was 400 lux. The compartments were connected by an opening (7.5 cm x 7.5 cm) located in the center floor of the divider. All experiments were performed between 19:00 and 21:00 during the dark period of the light-dark cycle. Each mouse was placed in the center of the white area for 10 minutes, and the number of times to switch between the two compartments and the time spent in the white area were recorded. The camera installed above the device allowed the behavior of the mouse to be observed on a video monitor in the adjacent room.
[0070]
The results obtained from the light-dark box revealed that the CRFR2 non-expressing mutant mice stayed in the light area of the light-dark box to the same extent as the control group, and moved between the light area and the dark area of the light-dark box (Fig. 4C and 4D). No significant difference was observed between the two groups in this experiment.
[0071]
Example 9
Effect of CRFR2 deletion on expression of other genes
Since no anatomical abnormalities were found in the components of the HPA axis as a whole (FIGS. 1D and 1E), changes in stress and behavioral responses in mutant mice resulted in altered gene expression of other components of the CRF signaling pathway. It is probably due to doing. To test for this possibility, the expression of UNC, CRF, and CRFR1 mRNA was examined by in situ hybridization.
[0072]
In situ hybridization was performed according to the method described above (15). Briefly, tissue sections (20 μm) were fixed with 4% paraformaldehyde, rinsed with PBS, immersed in acetic anhydride, dehydrated through a series of steps of ethanol, defatted with chloroform, and dehydrated again. . Then slide³⁵The S-labeled riboprobe was hybridized overnight in a 50% deionized formamide hybridization mixture at 55 ° C. in a humidified culture vessel. After incubation, the slides were washed with shaking in 1 × SSC at room temperature for 30 minutes, treated with 20 μg / ml RNase (Promega) at 37 C for 30 minutes, rinsed in 1 × SSC buffer at room temperature for 30 minutes, and shaken. Wash three times in 0.1X SSC at 65C for 20 minutes, rinse in 0.1X SSC at room temperature for 30 minutes, dehydrate with a series of stages of ethanol, air dry, Kodak hyperfilm (Eastman Kodak, Rochester, NY) ) For 3 days.
[0073]
After the film was developed, the slides were immersed in NTB2 liquid nuclear emulsion (Eastman Kodak; diluted 1: 1 with water), exposed for 10 days, processed for photography, counterstained with hematoxylin, and coverslipped. Slides were analyzed using the image analysis system Image Pro Plus (Media Cybernetics, Silver Springs MD). For analysis of PVN and cAmyg, the average optical density of each part was compared using the circle tool (area = 3022 pixels) to compare the part that matched the anatomical chart for each mouse to the same area of PVN and cAmyg. Judged. EW cell bodies expressing urocortin were too diffuse for analysis using standard optical density methods. Therefore, determine the number of cells in a designated EW that expresses a minimum optical density by color and cell size to pre-determine that the computer excludes non-positive cells and background silver particles. Such parameters were used. Thereafter, each cell determined positive by the computer for urocortin mRNA was counted to measure optical density. Thereafter, the average optical density and cell number were compared for each part.
[0074]
As illustrated in FIG. 5A, urocortin mRNA is expressed both in the number of expressing cells in mutant mice (FIG. 5B) and in urocortin mRNA concentration per cell (FIG. 5C) in the rostral region of the Eddinger Westphal (EW) nucleus. Significantly increased. Central nuclei of tonsils (cAmyg) showed a significant increase in CRF mRNA of non-expressing mutant mice (FIGS. 5A and 5D). No significant changes in CRF mRNA in PVN were seen in unstressed basal mice (FIGS. 5A and 5E). Expression patterns and levels of CRFR1 mRNA in the brain or anterior pituitary were not different between mutant and wild-type mice (not shown). These results indicate that CRFR2 non-expressing mutant mice had increased expression levels of CRF mRNA in cAmyg and urocortin mRNA in the rostral Edinger-Westphal nucleus.
[0075]
Example 10
Evaluation of hypotonia in response to UCN of CRFR2 non-expressing mutant mice
Previous studies have shown hypotonia in response to peripheral injections of urocortin (2). Furthermore, it has been hypothesized that CRFR2 is localized in vascular endothelial cells (3, 7) and is responsible for the vasodilator action of urocortin. To test this hypothesis, urocortin was injected into CRFR2 non-expressing mutant and control mice, and the blood pressure change of the mice was measured.
[0076]
The cardiovascular response to intravenous injection of urocortin and the vasodilator sodium nitroprusside was examined in mice anesthetized with isofluorine (wild type: n = 5; mutant: n = 3). The arterial catheter for recording blood pressure was softened and fabricated from disinfected PE-10 tubing stretched to an outer diameter of ~ 0.4 mm. The femoral artery was exposed and an arterial catheter infused with heparin saline (500 U / ml) was implanted and secured with surgical suture and tissue adhesive (Vetbond). The catheter was connected to a blood pressure transducer (Statham) and the arterial pressure pulse was displayed on a Gould pen recorder. Thereafter, a second catheter was implanted in the external jugular vein for intravenous injection of the drug. Drug infusion was performed for 30 minutes after completion of the cannulation procedure. The venous catheter was connected to a drug-filled syringe. The injection was completed within 0.5 to 1.0 minutes. Both wild-type and mutants received the same amount of urocortin (0.1 μg in 200 μl of 0.9% saline) and saline (as control).
[0077]
The dose used was determined from preliminary experiments citing data from corresponding studies in Sprague Dawley rats (2). To verify that the absence of a cerebrovascular response to urocortin injection in the mutant mouse was not due to the loss of the mouse's ability to vasodilate, the mutant mouse also recovered arterial pressure from urocortin injection, A second sodium nitroprusside injection (0.8 μg in 100 μl of 0.9% saline) was received. Mean arterial pressure (MAP) was determined from blood pressure tracking.
[0078]
Intravenous injection of urocortin (0.1 μg) resulted in a significant inhibitory response (−28.3 ± 2.0 mmHg) in control mice (FIG. 6). The reduction in arterial pressure persisted throughout the recorded period (90 to 120 minutes). In sharp contrast, the mutant mice did not show a measurable response to urocortin (only one mutant mouse showed a very small and transient reduction in pulse pressure (-3.5 mmHg). Is thought to be due to the injection pressure itself.) (FIG. 6). To verify that the peripheral vasculature of mutant mice allows vasodilation in response to different stimuli, nitroprusside sodium (NP), which causes vasodilation, was used as a nitric oxide donor in mutant mice. Administration. A rapid and strong inhibitory response in response to sodium nitroprusside injection (-30.0 ± 5.0 mmHg) was consistently observed.
[0079]
Example 11
Summary of the effects of CRFR2 deficiency on anxiety and stress
The results presented here suggest that CRFR2 non-expressing mutant mice exhibit a stress-sensitive and anxiety-like phenotype. Although basal feeding and weight gain were normal, the mutant mice responded to food deprivation by reducing food consumption after food deprivation stress. Although this may be a metabolic effect, the stress of food deprivation can alter the anxiety state of the mouse and reduce the appetite of the mouse. Mutant mice also show a rapid HPA response to restraint stress, again suggesting that these mice are more sensitive to stress. Since mutant mice showed higher and higher steroid levels than control mice, reduced ACTH levels after 10 minutes of restraint in mutant mice may also be a result of more rapid negative glucocorticoid feedback to the hypothalamus. Absent. In summary, although the HPA axis of mutant mice cannot rule out other physiological explanations for either a change in the feeding response or the rate of increase in response to stress, the feeding and HPA axis The results suggest that the CRFR2 non-expressing mutant mice are hypersensitive to stress.
[0080]
Mutant mice also show an increase in anxiety-like behavior within the EPM. However, these mice show similar levels of anxiety-like behavior in the light-dark box. Although pharmacological sensitivities and specificities are usually demonstrated in many animal studies on anxiety, different tasks are occasionally observed (16, 17). Both experiments are classified as unconditional search experiments, but the light-dark box measures neophobia in conjunction with the search. Behavior within the EPM is determined by searching for an aversion environment (18). Light conditions during the experiment also greatly affect the ability to detect anxiolytic or anxiety-producing effects in animal experiments. This aspect of the results for CRFR2 non-expressing mutant mice is not neophobia but indicates an enhanced affection related to the search for an aversive environment. Previous studies have shown a similar behavioral phenotype: neuropeptide Y (NPY) deficient mice are normal in the light-dark box but anxious in the EPM (19). These NPY mutant mice were classified as anxious and supported the previous finding that NPY injection reduced anxiety. The results obtained in CRFR2 non-expressing mutant mice indicate that EPM may be a more sensitive task for detecting anxiety in these mice.
[0081]
Example 12
Possible effects of increased CRF in cAmyg on anxiety
Since the amygdala expresses CRFR1 (7) and plays a major role in transducing stress signals (21), increased CRF in cAmyg resembles anxiety-like behavior and HPA axis hypersensitivity in mutant mice. Explain the increase in sex. In addition, the outer septum of the tonsil, which is rich in CRFR2, has been shown to regulate tonsil function (22-24), and impairment of the tonsil nucleus results in decreased ACTH secretion after restraint stress (25-28). Thus, during stress, CRFR2 in the lateral septum regulates tonsil function, and in the absence of CRFR2, leads to a rapid HPA response and increased anxiety-like behavior without impairment of tonsil function. Would.
[0082]
Tonsillar disorders have been shown to inhibit CRF-induced anxiety (21), and excessive affectiveness resulting from septal disorders (22). This neural pathway may explain the reduced anxiety-like behavior seen in CRFR1-deficient mice and the increased anxiety-like behavior in CRFR2-deficient mice (8). Thus, CRFR2 non-expressing mutant mice provide potential evidence for a novel mechanism of receptor regulation in anxiety-like behavior.
[0083]
Example 13
Possible mechanism of anxiety caused by increased UCN mRNA in the rostral EW
It has been shown that intravenous injection of urocortin induces behavior similar to anxiety, so an increase in urocortin mRNA in the rostral EW leads to an increase in behavior similar to anxiety in mutant mice. (29). Rostral EW injection into many regions of the CNS such as the locus coeruleus (LC) (30) and injection of the urocortin-related molecule CRF into locus coeruleus produces a response similar to anxiety (31) ). Thus, increased urocortin mRNA in the rostral EW activates locus coeruleus and enhances anxiety-like responses and / or hypersensitivity to stress.
[0084]
Example 14
Mutant mice not expressing CRFR2 and sensitivity of the autonomic nervous system
Additional explanations for increased anxiety-like behavior, such as increased autonomic nervous system sensitivity (32-34), cannot be ruled out yet. Previous studies using antisense oligonucleotides have found conflicting results regarding the role of CRFR2 in anxiety and behavior (35, 36).
[0085]
Although these studies show effects similar to anxiolytic effects of CRFR1 antisense oligonucleotide injection, none of the studies report consistent findings on CRFR2 antisense oligonucleotide injection. Although the technology of antisense oligonucleotide injection is potentially promising, it is currently being scrutinized because the reduced levels of protein cannot be replaced by complete removal of its target, as has been achieved in knockout mice.
[0086]
Example 15
Confirmation of the effect of UCN on vasodilation
The absence of CRFR2 in non-expressing mutant mice allowed confirmation of the effect of urocortin on vasodilation. Mutant mice did not respond at all to intravenous urocortin, whereas wild-type mice showed a dramatic decrease in mean arterial pressure. Nitroprusside injection results in vasodilation of mutant mice, and thus the lack of response to urocortin is not due to the physically non-dilatable vasculature of the mutant mice, but apparently to the lack of CRFR2. Was confirmed. These results indicate that the effect of urocortin on hypotension (2,14) occurs through its effect on CRFR2 in vascular endothelial cells, since CRFR2 non-expressing mutant mice showed no response to urocortin (3,7). Support temporary construction. Although the physiological stimuli most likely to cause UNC-induced vasodilation are currently unknown, the effects of urocortin on CRFR2 in the vasculature may be an interesting target in the development of antihypertensive drugs.
[0087]
wrap up
In summary, these results demonstrate that CRFR2-deficient mice display an anxiety-like behavior increase in the elevated plus maze and hypersensitivity of the HPA axis in response to stress. CRFR1 and CRFR2 non-expressing mutant mice provide a valuable model of anxiety and depression and may help to further elucidate the molecular mechanisms underlying these diseases. Studies of the CRF signaling pathway and its role in controlling anxiety and depression provide the necessary clues needed for effective treatment of these diseases.
[0088]
Example 16
Enhanced angiogenesis in CRFR2 non-expressing mutant mice
It was thought that CRFR2 non-expressing mutant mice exhibited increased blood vessel size and number in various tissues. Since the CRFR2 receptor and its activity were located in the endothelial cell layer of blood vessels (3,7), it was postulated that CRFR2 plays a role in regulating angiogenesis. To confirm that the CRFR2 non-expressing mutant mice had more large blood vessels, the tissues of the control group and the CRFR2 non-expressing mutant mice were treated with an antibody against the vascular specific marker platelet-endothelial cell-adhesion molecule (PECAM). Immunostaining was performed.
[0089]
Anterior pituitary gland, white adipose tissue, and tissue on the dorsal brain surface were obtained from both the 3-4 month old control group and the CRFR2 non-expressing mutant mouse. After fixing the tissue in 4% paraformaldehyde for 2 days, the tissue was exposed overnight in Dent's fix (methanol: DMSO 4: 1) with 5% hydrogen peroxide. The tissue is washed three times with 1% Tween-20 in 1 × TBS for 30 minutes each and diluted with 5% goat serum overnight in dilution buffer (0.5 M NaCl, 0.01 M PBS, 3.0% BSA). , And 0.3% Triton X-100) plus 1% DMSO at room temperature. The next day, a 1: 1000 dilution of anti-PECAM antibody (Pel Freeze) was added to the blocked mixture, followed by another 2 days at room temperature. The antibody was removed and the tissues were washed three times with 1X TBS plus Tween-20 and 1% DMSO for 1 hour each. Goat anti-RAT HRP secondary antibody was added at a dilution of 1: 5000 and incubated overnight at room temperature. The tissue was washed as above and the final wash was performed with 1 × TBS only for 1 hour at room temperature. The peroxidase reaction was performed in the presence of a reaction mixture containing glucose oxidase (Calbiochem) until a reddish brown color was formed. The tissue was dehydrated with a series of methanol stages and clarified with glycerol.
[0090]
The results of anti-PECAM immunostaining are shown in FIGS. 7A to 7F. According to these experiments, the absence of the CRFR2 receptor in non-expressing mutant mice caused an increase in the number and size of blood vessels in the anterior pituitary (FIG. 7B), white adipose tissue (FIG. 7D), and dorsal brain surface (FIG. 7F). It was confirmed to bring. The same tissue of the control mice is shown in FIG. 7A-anterior pituitary, FIG. 7C-white adipose tissue, and FIG. 7E-dorsal brain surface. Thus, one of the roles of the CRFR2 receptor in normal mice is to mediate CRF-induced inhibition of angiogenesis.
[0091]
Example 17
CRFR2 has no angiogenic effect on fetal mice
Anti-PECAM immunostaining experiments were performed on tissue sections from fetal mice 11 days after fertilization to determine whether the CRFR2 receptor was involved in angiogenesis during embryonic development. Tissue sections were made from fetal mice and processed in the same manner as sections from adult mice.
[0092]
FIG. 8A shows anti-PECAM immunostained sections from the heads of CRFR2 non-expressing mutant mice (right) and control mice (left), and FIG. 8B shows CRFR2-non-expressing mutant mice (right) and control mice (left). The immunostaining section from the forelimb is shown. No difference in the number or size of blood vessels was observed between the head or forelimb tissue sections of the CRFR2 non-expressing mutant mice and control mice. Therefore, CRFR2 is thought to be involved in angiogenesis only in fully grown mice.
[0093]
Example 18
Microfil-polymer characterization of angiogenesis in adult mice
To further characterize excessive angiogenesis in CRFR2 non-expressing mutant mice, the vascular tissues of control and mutant mice were perfused with microfil polymer to confirm that an increase in vascular volume occurred. In preparation for perfusion, a 30 standard needle was set in the left ventricle of anesthetized adult or 3-week-old CRFR2 non-expressing mutant mice and control mice. Perfusion was performed using a syringe pump until the irrigation was freely drained from the outlet in the right atrium for the purpose. Mice were placed at a temperature of 4 ° C. overnight to recover from the polymer. Tissue sections were removed from the recovered mice and dehydrated on day 1 via a series of graded ethanols beginning with 25% ethanol. After exposure to 6% hydrogen peroxide on the second day, 50% ethanol followed by 75% ethanol on the third day, 95% ethanol on the fourth day, and 100% ethanol on the fifth day. A series of ethanol treatments was continued. The tissue was clarified with glycerol before analysis. After removal of the soft tissue, the volume of the vascular bed of various tissues could be observed.
[0094]
FIGS. 9 and 10 show microfil-perfused tissues obtained from adult CRFR2 non-expressing mutant mice and control mice. In FIG. 9, tissues from normal mice are shown on the left side of each panel, and similar sections from CRFR2 non-expressing mutant mice are shown on the right. Increased numbers and sizes of blood vessels were observed in all tissues of CRFR2 non-expressing mutant mice, such as the dorsal brain surface (FIG. 9A), large intestine (FIG. 9B), and heart (FIG. 9C). In FIG. 10, the major arteries of the kidney (FIGS. 10A and 10B), the adrenal glands (FIGS. 10C and 10D), and the testes (FIGS. 10E and 10F) are indicated by arrows. The major blood vessels of CRFR2 non-expressing mutant mice (FIGS. 10B, 10D, and 10F) are significantly increased in size as compared to control mice (FIGS. 10A, 10C, and 10E). These results, combined with the results of anti-PECAM immunostaining, confirm that CRFR2-deficient mice show increased hypervascularization in all tissues observed, including brain, heart, pituitary, and gastrointestinal tract. Both the size and number of vessels increased.
[0095]
Microfil perfused tissues obtained from 3 week old mice are shown in FIGS. 11A-11D. CRFR2 non-expressing mutant mice show an increased number of blood vessels in the small intestine (FIG. 11B vs. 11A) and stomach (FIG. 11D vs. 11C). These results suggest that hypervascularization first increases the number of blood vessels and increases in the size of the blood vessels as the mice age.
[0096]
Example 19
Analysis of VEGF expression in CRFR2 non-expressing mutant mice
To determine whether CRFR2 affected vascular endothelial growth factor (VEGF) expression, tissues from CRFR2 non-expressing mutant mice and control mice were examined for VEGF content by Western blot analysis. White adipose tissue (WAT) and brown adipose tissue (BAT) were homogenized with buffer (50 mM TrisHCl, pH 7.4, 1 mM DRR, 2 mM MgCl2, 1 mM EDTA, 0.5 mM PMSF, 5 μg / ml leupeptin, 2 μg aprotinin). A 40 μg aliquot of the protein extract was separated on a 10% SDS-PAGE gel (Novex, San Diego) and transferred to a nitrocellulose membrane. Blots were blocked with 5% nonfat dry milk for 1 hour and washed with 1X TBS plus 0.2% Tween-20 (TBST). Thereafter, the blot was cultured for 1 hour with a 1: 1000 dilution of anti-VEGF antibody, washed twice with TBST for 20 minutes each, and cultured for 1 hour with a 1: 10,000 dilution of anti-rabbit HRP. After two washes of 20 minutes each with TBST, blots were visualized with ECL reagent. A representative blot is shown in FIG. Increased VEGF expression was observed in all tissues examined in CRFR2 non-expressing mutant mice, indicating a possible interaction between CRFR2 and VEBF production.
[0097]
Example 20
Promotion of hair growth in urocortin-treated mice
The skin in a small area of the mouse was shaved and a gel foam sponge impregnated with bFGF and urocortin, various growth factors and the CRF antagonist astresin was surgically implanted under the shaved skin. In mice implanted with both bFGF and urocortin, significant hair growth was observed just 5 days after the implanted sponge. Few hair growth was observed in the shaved area of mice implanted with sponges containing only growth hormone or astresine. FIG. 13 shows the hair growth of mice implanted with both urocortin and bFGF compared to mice implanted with bFGF alone. Thus, urocortin and bFGF stimulate rapid hair growth.
[0098]
The following references have been cited herein:
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2. Vaughan, J .; , Et al. , Nature 378, 287-292 (1995).
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7. Chalmers, et al. , J Neurosci 15, 6340-6350 (1995).
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9. Timpl, P .; et al. , Nat Genet 19, 162-166 (1998).
10. Webster, E.A. L. et al. , Endocrinology 137, 5747-5750 (1996).
11. Bornstein, S.M. R. et al. , Endocrinology 139, 1546-1555 (1998).
12. See Deak, T.W. et al. , Endocrinology 140, 79-86 (1999).
13. Spina, M .; et al. , Science 273, 1561-1564 (1996).
14． Schilling, et al. , Br J Pharmacol 125, 1164-1171 (1998).
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16. Hogg, S.M. Pharmacol Biochem Behav 54, 21-30 (1996).
17． Rodgers, R.A. J. Animal models of anxiety ': where next? Behav Pharmacol 8, 477-496; discussion 497-504 (1997).
18. Belzung, C .; & Le Pape, G.A. Physiol Behav 56, 623-628 (1994).
19. Palmiter, et al. , Recent Prog Home Res 53, 163-199 (1998).
20. See Heilig, et al. , Regul Pept 41, 61-69 (1992).
21. Liang, K.C. C. et al. , J Neurosci 12, 2313-2320 (1992).
22. King, F.S. A. & Meyer, P .; M. Effects of Amygdaloid Lections Upon Septal Hyperemotionality in the Rat. Science 128, 655-656 (1958).
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28. Marcilhac, et al. , Exp Physiol 81, 1035-1038 (1996).
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[0099]
All patents or publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. These patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.
[0100]
Those skilled in the art will immediately recognize that the present invention fulfills the stated objects and is well suited to the stated and attained goals and advantages inherent in the invention. This example is representative of, and is illustrative of, the presently preferred embodiments, in addition to the methods, procedures, treatments, molecules, and particular compounds described herein, which are intended to limit the scope of the invention. Not intended. Modifications and other uses of the invention which fall within the spirit of the invention as defined by the claims will be apparent to those skilled in the art.
[Brief description of the drawings]
In order that the above-listed features, advantages, and objects of the invention, and others set forth hereinafter, may be attained and will be understood in detail, the invention will be described in some embodiments with reference to the accompanying drawings. , And will be described more specifically. These drawings form part of the present specification. However, the accompanying drawings are only for illustrating preferred embodiments according to the present invention, and therefore should not be considered as limiting the scope of those embodiments.
FIG. 1A-1E
FIG. 4 shows the procedure used to generate CRFR2-deficient mice.
FIG. 1A
Genomic organization of the CRFR2 gene showing deletions of exons 10, 11, and 12, encoding from half of transmembrane region 5 to the end of transmembrane region 7. The figure also shows the target construct used for homologous recombination.
FIG. 1B
Disrupted alleles were detected by Southern blot analysis of tail DNA isolated from wild-type (+ / +), heterozygous (+/-), and non-expressing mutant (-/-) mice. FIG.
FIG. 1C
In CRFR2 control group (top) and mutant (bottom) mice¹²⁵FIG. 4 shows the autoradiographic binding of I-sorbazine. Note that there is no CRFR2 binding in the lateral septum of CRFR2 non-expressing mutant mice, but the cortical binding of CRFR1 is similar to that of control mice.
FIG. 1D
The figure which shows hematoxylin and eosin staining (H & E) of an adrenal gland. Note that there is no difference in the size of the adrenal gland at 10x magnification (top) and the structure of the adrenal gland at 20x magnification (bottom). C is cortex, M is medulla, ZG is globular band, ZF is bundled band, ZR is reticular layer, n = 8.
FIG. 1E
FIG. 9 shows H & E staining (upper panel) of adrenal glands sampled on liver for tissue sections at 4 × magnification, n = 8. Pituitary adrenocorticotropic factors were identified using anti-ACTH antibody at a magnification of x10, n = 5 (20) (lower panel). P is posterior lobe, I is middle lobe, A is anterior lobe. No anatomical differences were observed in the localization or expression level of the adrenal cortex stimulating factor of the adrenal gland or ACTH.
FIG. 2A-D
The figure which shows the hypersensitivity of the HPA axis | shaft with respect to the stress of a mutant animal. * = Significantly different from wild type control group at the same time point, p <0.01 by Sheffee post hoc test. Plasma obtained by unanesthetized retro-orbital bleeding.
FIG. 2A
FIG. 7 shows pre-stress ACTH plasma levels at 7:00 AM. n = 16.
FIG. 2B
FIG. 7 shows basal levels of corticosterone plasma at 7:00 AM and 5:00 PM. n = 5.
FIG. 2C
The figure which shows the time course of the suppression stress effect with respect to ACTH.
FIG. 2D
FIG. 9 shows that corticosterone plasma levels (7:00 AM) are significantly different from the wild-type control group at the same time point. n = 7.
FIG. 3A-B
The figure which shows the effect which 24 hours of food deprivation has on feeding in a wild type and a mutant littermate mouse.
FIG. 3A
FIG. 4 shows the basal food consumption of mutant mice (n = 7) compared to wild-type littermate mice (n = 10) and the food consumption immediately after a 24 hour food deprivation period. P <0.001 by Scheffe post hoc test.
FIG. 3B
The figure which shows the basal body weight (open bar line) of a wild type and a mutant type | mold mouse | mouth, and the body weight (black bar line) when 24 hours passed after re-feeding after 24 hours of food deprivation. Note that there is no difference in basal weight and body weight after refeeding between experimental groups.
FIGS. 4A-4D
The figure which shows that the behavior similar to anxiety of a mutant animal increases in an elevated plus maze.
(Control group n = 7, mutant type n = 7; mean ± SEM)
FIG. 4A
The figure which shows the percentage (**, p <0.005) of the staying time in an open arm, and the frequency | count (*, p <0.02) which visited the open arm. All mutant mice were significantly less than the wild-type control group.
FIG. 4B
The figure which shows the locomotor activity measured by the total approach (p = 0.64) of the approach to a closed arm, and the total approach (p = 0.38) of an arm. There was no difference between the control group and the mutant animals.
FIG. 4C
There was no difference in anxiety-like behavior measured in the light-dark box experiment for the time spent in the bright part of the box.
FIG. 4D
There were no differences in anxiety-like behaviors measured in the light-dark box experiment with respect to the number of movements between the light and dark parts of the box.
5A to 5E.
The figure which shows that the level of urocortin and CRF mRNA increased in the mutant brain. From 4B to 4E, n = 3, ± SEM, *, p <0.05, **, p <0.01, ***, p <0.005.
FIG. 5A
Shown are silver particles resulting from in situ hybridization (23) to urocortin mRNA in the rostral EW (top) at 20 × magnification and cAmyg (middle) and CRF mRNA in the paraventricular nucleus (bottom) at 10 × magnification. FIG.
FIG. 5B
The figure which shows the result of having determined the number of cells which express urocortin mRNA in the rostral EW using semi-quantitative analysis of silver particles.
FIG. 5C
The figure which shows the average light density of urocortin mRNA per one cell.
FIG. 5D
The figure which shows the optical density of CRF mRNA in cAmyg.
FIG. 5E
The figure which shows the optical density of CRF mRNA in a paraventricular nucleus.
FIG. 6
The figure which shows the cardiovascular responsiveness to the intravenous injection of 1.0 microgram of urocortin in a wild type (n = 5) and a mutant type | mold mouse (n = 3). Note that there is no significant suppressive response of mutant mice to urocortin injection. ***, p <0.005.
7A to 7F.
Figures showing tissues from adult CRFR2 control groups (Figures 7A, 7C, and 7E) and CRFR2 non-expressing mutant (Figures 7B, 7D, and 7F) mice immunostained with anti-PECAM antibody. These studies show that the anterior pituitary gland (FIG. 7B), white adipose tissue (FIG. 7D), and dorsal brain surface (FIG. 7F) of the control mice have anterior pituitary gland (FIG. 7A), Increased number and size of vessels were shown as compared to adipose tissue (FIG. 7C) and dorsal brain surface (FIG. 7E).
FIG. 8A-8B.
The figure which shows the immunostained structure | tissue obtained from the embryonic body CRFR2 non-expression mutant mouse and the control group mouse on the 11th day after fertilization. FIG. 8A shows tissues obtained from the heads of CRFR2 non-expressing mutant mice (right) and control mice (left). FIG. 8B shows tissues obtained from the forefoot of the CRFR2 non-expressing mutant mouse (right) and the control group mouse (left). No difference in the number and size of vessels in either the head or the forefoot
9A to 9C.
FIG. 9 shows microfil perfused tissues obtained from adult CRFR2 non-expressing mutant mice (right, FIGS. 9A, 9B, and 9C) and control mice (left, FIGS. 9A, 9B, and 9C). CRFR2 non-expressing mutant mice show increased vasculature on the dorsal brain surface (FIG. 9A), large intestine (FIG. 9B), and heart (FIG. 9C).
10A to 10F
FIG. 10 shows microfil-perfused tissues from adult CRFR2 non-expressing mutant mice (FIGS. 10B, 10D, and 10F) and control mice (FIGS. 10A, 10C, and 10E). Arrows indicate the major arteries of the kidney (FIGS. 10A and 10B), adrenal glands (FIGS. 10C and 10D), and testes (FIGS. 10E and 10F).
11A to 11D.
FIG. 12 shows microfil-perfused tissues obtained from CRFR2 non-expressing mutant mice (FIGS. 11B and 11D) and control mice (FIGS. 11A and 11C) three weeks after birth. Mutant mice show increased numbers of vessels in the small intestine (FIG. 11B vs. 11A) and stomach (FIG. 11D vs. 11C).
FIG.
FIG. 7 shows a Western blot demonstrating increased VEGF expression in white adipose tissue (WAT) and brown adipose tissue (BAT) from CRFR2 non-expressing mutant mice.
FIG. 13
FIG. 2 shows that surgical implantation of a gel foam sponge impregnated with urocortin and bFGF promoted hair growth in the area directly covered by the sponge implant. The right mouse was treated with a sponge containing only bFGF. The left mouse was implanted with a sponge impregnated with both urocortin and bFGF.

Claims (27)

A non-natural transgenic mouse in which at least one allele of corticotropin releasing factor receptor 2 (CRFR2) has been disrupted such that the allele does not express corticotropin releasing factor receptor 2 (CRFR2) protein. The transgenic mouse according to claim 1, wherein the DNA base sequences of exons 10, 11, and 12 of the corticotropin releasing factor receptor 2 allele have been removed. The transgenic mouse according to claim 2, wherein the DNA base sequence is replaced with a neomycin resistance gene cassette. 4. The transgenic mouse of claim 3, wherein said mouse is heterozygous for said replacement. 4. The transgenic mouse of claim 3, wherein said mouse is homozygous for said replacement. 4. Progeny of a cross between the mouse of claim 3 and a mouse of another strain. In a method of screening for a compound having anxiolytic activity,
a) administering the compound to the transgenic mouse according to claim 5;
b) testing the mouse for behaviors associated with anxiety; and c) mimicking anxiety in the second transgenic mouse of claim 5 wherein the compound is not administered. A method comprising the steps of comparing with the action taken. The method of claim 7, wherein the mouse is tested for anxiety in an elevated plus maze. In a method of screening for a compound having a depressive alleviating activity,
a) administering the compound to the transgenic mouse according to claim 5;
b) testing the mouse for depression-like behavior and c) depressing the mouse-like behavior of the second transgenic mouse according to claim 5, wherein the compound is not administered. A method consisting of comparing steps. In a method for screening a compound that regulates blood pressure,
a) administering the compound to the transgenic mouse according to claim 5;
b) testing the blood pressure change of said transgenic mouse; and c) comparing the blood pressure change of said transgenic mouse to the blood pressure change of a second mouse, wherein said second mouse is administered with said compound. A method comprising selecting from the group consisting of the transgenic mouse according to claim 5, which was not performed, and a wild-type mouse to which the compound was also administered. In a method of screening for a compound that affects angiogenesis,
a) administering the compound to the transgenic mouse according to claim 5;
b) assessing the change in angiogenesis of the transgenic mouse; and c) comparing the change in angiogenesis of the transgenic mouse with the compound without administering the compound. Comparing the angiogenic changes in a mouse selected from the group consisting of the administered wild-type mice. A method of screening for a compound that has an effect on the response of the hypothalamus-pituitary-adrenal axis to stress,
a) administering the compound to the transgenic mouse according to claim 5;
b) placing the mouse in a stress-induced situation;
c) monitoring the plasma levels of corticosterone and adrenocorticotropic hormone in the mouse; and d) comparing the levels to those in the transgenic mouse of claim 5, which are not placed in the stress-induced situation. Method. The method of claim 12, wherein the stress-inducing situation is a physical restraint stress. In a method for determining the effect of CRFR2 on a second protein,
a) administering an agent (agonist) affecting the second protein to the transgenic mouse according to claim 5;
b) evaluating the second protein, wherein the evaluation is selected from the group consisting of evaluation of protein expression and evaluation of protein activity; and c) evaluation results for the transgenic mouse. Comparing the results with those obtained from wild-type mice to which the agent has been administered. 15. The method of claim 14, wherein said second protein is selected from the group consisting of corticotropin releasing factor, corticotropin releasing factor receptor 1, urocortin, corticotropin receptor and urocortin receptor. A method of enhancing angiogenesis in a target tissue, comprising administering a CRFR2 antagonist to said target tissue. 17. The method of claim 16, wherein the CRFR2 antagonist is an antisense nucleotide directed against CRFR2. 17. The method of claim 16, wherein said target tissue is selected from the group consisting of heart, brain, pituitary, gonads, kidney, fat, and gastrointestinal tissue. 17. The method of claim 16, wherein said angiogenesis is enhanced in an individual having a pathological condition selected from the group consisting of infarction, stroke, and injury. A method for inhibiting angiogenesis in a target tissue, comprising administering a CRFR2 agonist to the target tissue. 21. The method of claim 20, wherein said CRFR2 agonist is selected from the group consisting of urocortin and CRF. 21. The method of claim 20, wherein said tissue is selected from the group consisting of heart, brain, pituitary, gonads, kidney, fat, and gastrointestinal tissue. 21. The method of claim 20, wherein said angiogenesis is suppressed in an individual having a pathological condition selected from the group consisting of cancer and diabetic retinopathy. A method of promoting hair growth comprising contacting urocortin with a skin area where hair growth is desired. The method of claim 24, wherein the urocortin is implanted under the skin. 25. The method of claim 24, wherein bFGF is administered to the skin before, after, or simultaneously with urocortin. 25. The method of claim 24, wherein urocortin is included in the composition with bFGF.

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